DNA constructs and plants incorporating them

ABSTRACT

Expression of genes inserted into plants by transformation is controlled by the use of a promoter selected from Aco1, Aco2 and Aco3, the sequences of which are given. The level of expression obtained by use of these promoters varies with the stage of development of the plant.

The present invention relates to DNA constructs and plants incorporatingthem. In particular, it relates to promoter sequences for the expressionof genes in plants.

Gene expression is controlled by various regulatory components,including nucleic acid and protein elements. In particular, geneexpression is controlled by a region commonly referred to as the“promoter” which lies upstream (5′) of the protein encoding region. Apromoter may be constitutive or tissue-specific,developmentally-regulated and/or inducible.

Within the promoter region there are several domains which are necessaryfor full function of the promoter. The first of these domains liesimmediately upstream of the structural gene and forms the “core promoterregion” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence which defines thetranscription start point for the structural gene. The precise length ofthe core promoter region is indefinite but it is usuallywell-recognisable. Such a region is normally present, with somevariation, in all promoters. The base sequences lying between thevarious well-characterised “boxes” appear to be of lesser importance.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

Manipulation of crop plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation therefore relies on the availability of means to drive andto control gene expression as required; for example, on the availabilityand use of suitable promoters which are effective in plants and whichregulate gene expression so as to give the desired effect(s) in thetransgenic plant. It is advantageous to have the choice of a variety ofdifferent promoters so that the most suitable promoter may be selectedfor a particular gene, construct, cell, tissue, plant or environment.

Promoters (and other regulatory components) from bacteria, viruses,fungi and plants have been used to control gene expression in plantcells. Numerous plant transformation experiments using DNA constructscomprising various promoter sequences fused to various foreign genes(for example, bacterial marker genes) have led to the identification ofuseful promoter sequences. It has been demonstrated that sequences up to500-1000 bases in most instances are sufficient to allow for theregulated expression of foreign genes. However, it has also been shownthat sequences much longer than 1 kb may have useful features whichpermit high levels of gene expression in transgenic plants. A range ofnaturally-occurring promoters are known to be operative in plants andhave been used to drive the expression of heterologous (both foreign andendogenous) genes in plants: for example, the constitutive 35Scauliflower mosaic virus promoter, the ripening-enhanced tomatopolygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology,11:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO,7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989,Plant Molecular Biology, 13:639-651) and many others.

As stated above, successful genetic manipulation relies on theavailability of means to control plant gene expression as required. Thescientist uses a suitable expression cassette (incorporating one or morepromoters and other components) to regulate gene expression in thedesired manner (for example, by enhancing or reducing expression incertain tissues or at certain developmental stages). The ability tochoose a suitable promoter from a range of promoters having differingactivity profiles is thus important.

One object of the present invention is to provide alternative promoterscapable of driving gene expression in plants. Such promoters aresuitable for incorporation into DNA constructs encoding any target geneso that the target gene is expressed when the construct is transformedinto a plant. It may be particularly advantageous to provide alternativepromoters which exhibit particular spatial or temporal patterns ofexpression, for example promoters which are active in certain cell-typesand/or are particularly responsive to certain developmental events andenvironmental conditions. This may allow more selective control of geneexpression and its effects, as the target gene is only activated whereand/or when it is required.

In work leading to the present invention, we have isolated and fullysequenced three ACC oxidase gene promoters from tomato. ACC oxidase isan enzyme involved in the biosynthesis of ethylene.

Ethylene is a major plant hormone which has been shown to have a varietyof effects on plant growth and development in many species. Endogenouslevels of ethylene increase during several stages of development and inresponse to various stimuli including mechanical wounding and pathogeninfection, ripening of climacteric fruits and leaf and flowersenescence. The biosynthetic pathway for ethylene in plants iswell-established; for example, a review of ethylene biosynthesis waspublished by Yang and Hoffman in 1984 (Annual Review Plant Physiology,35:155-189). The final stages of ethylene biosynthesis proceed by thefollowing pathway:

Methionine→

S-adenosyl-L-methionine (SAM)→

1-aminocyclopropane-1-carboxylic acid (ACC)→Ethylene.

The final step in the pathway of ethylene biosynthesis is the conversionof the cyclic amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) toethylene. This reaction is catalysed by the enzyme ACC oxidase (alsoknow as ethylene forming enzyme or EFE) which was once thought to beconstitutively expressed in most tissues. However, since the cloning ofthe gene the messenger RNA has been shown to be induced under a numberof conditions known to result in increased ethylene production (Daviesand Grierson, 1989, Planta, 179:73-80; Hamilton et al, 1990, Nature,346:284-287).

In tomato, ACC oxidase is encoded by a multigene family comprising threemembers, hereinafter called the Aco1 gene, the Aco2 gene and the Aco3gene. Bouzayen et al (1993, pp 76-81 in Cellular and molecular aspectsof the plant hormone ethylene, eds. Pech et al, Kluwer AcademicPublishers, NL) discuss the expression and characterisation of the ACCoxidase (EFE) multigene family in tomato plants, and FIG. 1 shows thestructure and similarity of the gene family. When the open reading frameregions of the three tomato ACC oxidase (Aco) genes are aligned, theoverall identity is 79.3%. The 5′ or 3′ un-translated regions are lesshomologous than the coding regions.

The coding region of the Aco1 tomato gene corresponds to the TOM13 cDNAclone, first described as a ripening-related clone by Slater et al(1985, Plant Molecular Biology, 5:137-147).

The coding region of the Aco2 tomato gene corresponds to the gTOMA genesequence published by Holdsworth et al in Nucleic Acids Research, 1987,15:10600.

The Aco3 tomato gene is equivalent to the clone gTOMB, described(without sequence data) in Holdsworth et al, 1988, Plant MolecularBiology, 11:81-88. Because no expression of gTOMB (Aco3) was detected,Holdsworth et al suggested this was a pseudogene, thus suggesting thatan active and useful promoter could not be isolated from this gene.

After the cloning of the first ACC oxidase cDNA clone (pTOM13), standardhybridisation procedures were used to isolate clones for ACC oxidasefrom other plant species. ACC oxidase cDNA or genomic clones have nowbeen isolated from at least nine other species:

(1) Melon (Cucumis melo)

Balague et al, 1993, Eur J Biochem, 212:27-34;

(2) Petunia (Petunia hybrida)

Wang and Woodson, 1992, Plant Physiol, 100:535-536;

(3) Apple (Malus domestica)

Ross et al, 1992, Plant Molecular Biology, 19:231-238;

(4) Mustard (Brassica juncea)

Pua et al, 1992, Plant Molecular Biology, 19;541-544;

(5) Avocado (Persea americana)

Christofferson et al, 1993, Cellular and molecular aspects of the planthormone ethylene, Pech J C et al (eds), Kluwer, pages 65-71;

(6) Peach (Prunus persica)

Callahan et al, 1992, Plant Physiol, 100:482-488;

(7) Orchid (Phalaenopsis)

Nadeau et al, 1993, Plant Physiol, 103:31-39;

(8) Kiwifruit (Actinidia deliciosa)

Macdiarmid and Gardiner, 1993, Plant Physiol, 101:691-692;

(9) Carnation (Dianthus caryophyllus)

Wang et al, 1991, Plant Physiol, 96:1000-1001.

The whole or part of the protein coding regions of ACC oxidase genes maybe incorporated into DNA constructs for plant transformation.International patent application publication number WO91/01375 describesa method of modifying ethylene biosynthesis in plants by using DNAconstructs based on genes encoding an enzyme involved in ethylenebiosynthesis (such as ACC oxidase). Sense constructs as well asantisense constructs may be used to regulate gene/enzyme activity.

We have now isolated and fully sequenced three ACC oxidase genepromoters from tomato, and have characterised the activity andexpression patterns of these promoters. Such promoters may be used todrive the expression of target genes encoded by DNA constructs withintransgenic plants.

According to the present invention, there is provided a DNA sequenceencoding a gene promoter capable of driving gene expression in plantswhich is selected from the group consisting of the Aco1 gene promoterhaving the sequence shown as SEQ ID NO: 1 or active variants thereof,the Aco2 gene promoter having the sequence shown as SEQ ID NO: 2 oractive variants thereof, and the Aco3 gene promoter having the sequenceshown as SEQ ID NO: 3 or active variants thereof. “Active variants” areDNA sequences homologous to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3which retain promoter activity.

The nucleotide sequence of the Aco1 promoter is shown as SEQ ID NO: 1(1925 bases). The ATG start codon is shown at the end of the promotersequence (base number 1923 to 1925). The putative TATA-box is betweenbase number 1797 and base number 1802. The transcriptional start site isat base number 1826 (for the Aco1 gene promoter, the naturally-occurringdownstream coding sequence is TOM13). The Aco1 promoter was isolated byinverse PCR as it could not be cloned by conventional procedures.

The nucleotide sequence of the Aco2 promoter is shown as SEQ ID NO: 2(2510 bases). The ATG start codon is shown at the end of the promotersequence (base number 2508 to 2510). The putative TATA-box is betweenbase number 2357 and base number 2362. The transcriptional start site isat base number 2386. The genomic clone for Aco2 was isolated from atomato genomic DNA library using the TOM13 cDNA clone as a probe. TheAco2 (gTOMA) gene sequence published by Holdsworth et al, 1987 (NucleicAcids Research, 15:10600) included a very limited promoter region(approximately 350 base pairs) starting at base number 2083 in SEQ IDNO: 2. We now provide over 2 kb of additional sequence derived from twoEcoRI fragments (1.3 kb and 1.6 kb) of the same λ-clone.

The nucleotide sequence of the Aco3 promoter is shown as SEQ ID NO: 3(2483 bases). The ATG start codon is shown at the end of the promotersequence (base number 2481 to 2483). The putative TATA-box is betweenbase number 2370 and base number 2375. The transcriptional start site isat base number 2404. The genomic clone for Aco3 was isolated from atomato genomic DNA library using the TOM13 cDNA clone as a probe. Wehave shown that the Aco3 (gTOMB) gene is not a pseudogene as had beensuggested in Holdsworth et al, 1988 (Plant Molecular Biology, 11:81-88).The Aco3 gene and promoter are contained in the 4.2 kb genomic insert ofclone gTOMB. We have now sequenced the 2.4 kb upstream sequence thatacts as a promoter to direct Aco3 gene expression. Having completelysequenced the Aco3 gene, we found the coding region (but not promoter)is homologous to the sequence of the cDNA clone pHTOM5 published bySpanu et al, 1991, EMBO J, 10:2007-2013 (a clone expressed in culturedcells in response to treatment with fungal elicitor).

The sequences of the Aco1, Aco2 and Aco3 gene promoters have notpreviously been elucidated. Example 1 gives information on the limitedhomology between the Aco promoters and known promoters.

The Aco promoters may be synthesised ab initio using the sequences shownin SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 as a guide.Alternatively, the Aco2 and Aco3 promoters may be isolated from plantgenomic DNA libraries using suitable probes derived from the saidsequences and the Aco1 promoter may be isolated using a PCR approach.

Active variants of the Aco promoters may also be generated. It may bepossible to alter the level or type of activity of the Aco promoters bymanipulating their sequences: for example, by altering the nucleotidesequence in key regulatory regions, by truncating the sequence or bydeleting parts within the sequence. Segments of the Aco promotersequences of between 100 and 2000 bases in length may be useful asplant-operative promoters.

Gene specific probes were generated from the 3′ UTR region of each ofthe Aco1, Aco2 and Aco3 genes. These were used in conjunction with aribonuclease protection assay (Example 2) to analyse the differentialexpression of the Aco genes in various tissues and in response todifferent stimuli known to induce ethylene biosynthesis. Theseexperiments have indicated where and when the Aco genes are expressed(that is, where and when the Aco promoters are active). ACC oxidase geneexpression is highly regulated. Different genes are expressed in varioustissues in response to different stimuli and the messages accumulate todifferent levels. ACC oxidase genes are highly inducible and thereforenot constitutively expressed. There may also be post-transcriptionalregulation of the Aco genes.

The Aco1 promoter is the strongest Aco promoter, being stronglyexpressed during ripening and in response to wounding. It may be usefulfor expressing genes during ripening or in response to wounding (forexample, by driving “defense” genes) or for controlling expression ofantisense constructs. The Aco1 gene and its encoded product has a likelyrole in the senescence of leaves, flowers and fruit. The Aco1 promotermay be useful for driving expression of target genes used to modify thesenescence process in plants.

The Aco2 gene is expressed in seedlings and flowers. The Aco2 promotercould have utility in specific circumstances or cell types.

The Aco3 gene is expressed during ripening and in response to wounding,although less strongly than Aco1. Aco3 may act as the trigger to induceAco1 during autocatalytic ethylene production that occurs during fruitripening and flower senescence.

All the Aco genes appear to play a role in postpollination events intomato flowers and may also be important for root development duringgermination.

In practice the promoter of the invention may be inserted as a promotersequence in a recombinant gene construct destined for use in a plant.The construct is then inserted into the plant by transformation. Anyplant species may be transformed with the construct, and any suitabletransformation method may be employed.

According to a second aspect of the invention, there is provided a plantgene expression cassette comprising a promoter operatively linked to atarget gene, the promoter being selected from the group consisting ofthe Aco1 gene promoter having the sequence shown as SEQ ID NO: 1 oractive variants thereof, the Aco2 gene promoter having the sequenceshown as SEQ ID NO: 2 or active variants thereof, and the Aco3 genepromoter having the sequence shown as SEQ ID NO: 3 or active variantsthereof.

The target gene is a DNA sequence which may be derived from anendogenous plant gene or from a foreign gene of plant, fungal, algal,bacterial, viral or animal origin. Normally it is a sequence other thanthe sequence encoding the ACC oxidase protein which follows the Acopromoter in the naturally-occuring Aco gene. The target gene may be asingle gene or a series of genes. The target gene is adapted to betranscribed into functional RNA under the action of plant cell enzymessuch as RNA polymerase. Functional RNA is RNA which affects thebiochemistry of the cell: for example, it may be mRNA which istranslated into protein by ribosomes or it may be RNA which inhibits thetranslation of mRNA related to it. Thus the target gene sequence may bea sense sequence encoding at least part of a functional protein or anantisense sequence.

The expression cassette is suitable for general use in plants. Inpractice the DNA construct comprising the expression cassette of theinvention is inserted into a plant by transformation. Any transformationmethod suitable for the target plant or plant cells may be employed,including infection by Agrobacterium tumefaciens containing recombinantTi plasmids, electroporation, microinjection of cells and protoplasts,microprojectile transformation, pollen tube transformation andtransformation of plant cells using mineral fibres (U.S. Pat. No.5,302,523, International Patent Application Publication NumberWO94/28148). The transformed cells may then in suitable cases beregenerated into whole plants in which the new nuclear material isstably incorporated into the genome. Both transformed monocotyledonousand dicotyledonous plants may be obtained in this way. Transgenic planttechnology is for example described in the following publications: SwainW F, 1991, TIBTECH 9: 107-109; Ma J K C et al, 1994, Eur J Immunology24: 131-138; Hiatt A et al, 1992, FEBS Letters 307:71-75; Hein M B etal, 1991, Biotechnology Progress 7: 455-461; Duering K, 1990, PlantMolecular Biology 15: 281-294.

Examples of genetically modified plants which may be produced includebut are not limited to field crops, cereals, fruit and vegetables suchas: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat,barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears,strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage,onion.

The invention further provides a plant cell containing a gene expressioncassette according to the invention. The gene expression cassette may bestably incorporated in the plant's genome by transformation. Theinvention also provides a plant tissue or a plant comprising such cells,and plants or seeds derived therefrom.

The invention further provides a method for controlling plant geneexpression comprising transforming a plant cell with a plant geneexpression cassette having an Aco promoter operatively linked to atarget gene, whereby the activated promoter drives expression of thetarget gene. The promoter may be activated under certain spatial,temporal, developmental and/or environmental conditions.

In order to determine their temporal and spatial expression, thepromoter fragments of the Aco genes are fused to the GUS(β-glucuronidase) reporter gene in DNA constructs suitable for planttransformation. GUS accumulation in transgenic plants may then bemonitored. Example 3 describes some of these experiments. Transgenicplants expressing a GUS reporter gene under control of the Aco1 promoterhave been generated. Analysis has shown that GUS is expressed in woundedtissue (such as leaves) and ripening fruit (Example 3). Transgenicplants expressing a GUS reporter gene under control of the Aco2 promoteror the Aco3 promoter may also be generated for analysis.

The invention will now be described by way of example only withreference to the accompanying drawings in which:

FIG. 1 is a diagram showing the structure and similarity of the tomatoACC oxidase gene family;

FIG. 2 is a diagram of the Aco1 promoter/GUS transformation constructs;

FIG. 3 is a diagram of the Aco3 promoter/GUS transformation constructs.

The invention is also described with reference to the SEQUENCE LISTINGin which:

SEQ ID NO: 1 shows the nucleotide sequence of the Aco1 promoter;

SEQ ID NO: 2 shows the nucleotide sequence of the Aco2 promoter;

SEQ ID NO: 3 shows the nucleotide sequence of the Aco3 promoter;

SEQ ID NO: 4 shows the nucleotide sequence of the TCA-motif;

SEQ ID NO: 5 shows the nucleotide sequence of the GCCGCC-motif tobacco

SEQ ID NO: 6 shows the nucleotide sequence of the H-box from a range ofinducible promoters;

SEQ ID NO: 7 shows the nucleotide sequence of the H-box from the Aco3promoter.

EXAMPLE 1 Analysis of the Aco1, Aco2 and Aco3 Gene Promoter Sequences

(a) Homology searches

Homology searches with the EMBL database using the BLASTN and BESTFITalgorithms for the promoters of the tomato Aco1, Aco2 and Aco3 genesgave the following results.

Aco1 promoter

tomato 2A11 gene

(Van Haren and Houck, 1991, PMB, 17:615-630):

87% identity in 511 bp overlap

tomato E4 gene

(Cordes et al, 1989, Plant Cell, 1:1025-1034):

84.5% identity in 413 bp overlap

petunia (Ph) ACC oxidase genes

(Tang et al, 1993, PMB, 23:1151-1164):

Ph-Aco1:

70% identity in 249 bp overlap

nt −728 to −563 (Ph-Aco1)

nt −369 to −134 (Le-Aco1)

Ph-Aco3:

74% identity in 330 bp overlap comprising the region at and before theTATA-box

Ph-Aco4:

57% identity in 118 bp overlap comprising TATA-box and 5′UTR in bothgenes

Aco2 promoter

petunia ACC oxidase genes:

homologies in 5′UTR and around TATA-boxes with all 3 genes

β-1,3 glucanase genes

(Linthorst et al, 1990, PNAS, 87:8756-8760) and chitinase genes

(Broglie et al, 1989, Plant Cell, 1:599-607) from tobacco and broadbean: short stretches (60-70% identity in 70-100 bp overlaps)

Aco3 promoter:

potato WTN2 gene

(Stanford et al, 1989, MGG, 215:200-208):

85% identity in 157 bp overlap

nt −2384 to −2226 (Aco3)

nt −289 to −132 (WIN2)

petunia ACC oxidase genes:

Ph-Aco1:

68% identity in 57 bp overlap

nt −434 to −378 (Le-Aco3)

nt −682 to −626 (Ph-Aco1)

Ph-Aco3:

86% identity in 38 bp overlap around TATA-box

Ph-Aco4:

74% identity in 54 bp overlap

nt −534 to −480 (Le-Aco3)

nt −417 to −367 (Ph-Aco4)

poplar proteinase inhibitor gene

(Bradshaw et al, 1990, PMB, 14:51-59):

70% identity in 57 bp overlap

tobacco PR1 gene

(Oshima et al, 1987, FEBS Lett, 225:243-246):

62% identity in 80 bp overlap around TATA-box

(b) Occurrence of cis-acting elements

The sequences of the tomato Aco gene promoters were searched for certaincis-acting elements (boxes) known from ethylene/wound/elicitorresponsive genes.

The TCA-motif “TCATCTTCTT” (SEQ ID NO: 4) is a 10 bp motif which occursin over thirty stress and pathogen inducible promoters (Goldsborough etal, 1993, Plant J, 3:563-571) and is bound by tobacco nuclear proteins.It occurs seven times in the tomato Aco1 gene promoter (2 mismatches),five times in Aco2 (1 and 2 mismatches) and eight times in Aco3 (1 and 2mismatches).

The GCCGCC-motif is found in several ethylene induced PR genes fromtobacco which contain a highly conserved 11 bp element “TAAGAGCCGCC”(SEQ ID NO: 5). Part of this is present in a bean chitinase minimalethylene response element. It is presumed not to occur inripening/senescence related ethylene responsive genes (Eyal et al, 1993,Plant J, 4:225-234; Meller et al, 1993, PMB, 23:453-463; Hart et al,1993, PMB, 21:121-131). It is found in Aco3 at nucleotide (nt) −1895,and in Aco1 and Aco2 only with 1 mismatch.

There is an 8 bp element in the carnation GST1 promoter (“ATTTCAAA”)(Itzhaki et al, 1994, PNAS, 91:8925-8929) and the tomato E4 promoter(“AATTCAAA”) (Montgomery et al, 1993, PNAS, 90:5943) in regionsnecessary for ethylene response, bound by nuclear proteins fromsenescing petals/unripe fruit. The element occurs three times in thetomato Aco1 gene, twice in Aco2 and twice in Aco3.

The H-box “CCTACC(N)₇CT” (SEQ ID NO: 6) occurs in a range of induciblepromoters responsive to various stimuli (ABA, light, UV irradiation,elicitors) (Dixon et al, 1988, Ann Rev Phytopathol, 32:479-502). Itoccurs as “CATACC(N)₇CT” (SEQ ID NO: 7) at nucleotide −1383 in Aco3 butnot in Aco1 or Aco2.

EXAMPLE 2 Ribonuclease Protection Assays: Analysis of the DifferentialRegulation of the ACC Oxidase Gene Family from Tomato

(a) Method

To study ACC oxidase gene expression, mRNA was extracted from varioustissues in ripening tomato fruit, in ethylene treated fruit, ingerminating seeds and in flowers at various stages of development (stage1, closed buds; stage 2, buds begining to open but petals green; stage3, fully open flowers; stage 4, early senescent flowers—curled but notfaded petals; stage 5, late senescent flowers—both petals and sepalscurled and petals turning brown). Total RNA was extracted from plants asdescribed in Hamilton et al, 1990, Nature, 346:284-287 and 25 μg wasused routinely in each ribonuclease protection assay.

Radiolabelled (P^(32*)) RNA probes were generated from linearisedrecombinant plasmids containing the 3′ UTR region of each Aco gene usingT7 RNA polymerase (Promega) as outlined in the Promega protocols andapplications handbook. Each probe was gene-specific so that there wouldbe no cross-hybridisation between the Aco1, Aco2 or Aco3 probes/genes.Probes were purified by separation through denaturing 8% PAGE gels.After visualisation of the full length probe by autoradiography the bandwas excised from the gel and the probe eluted overnight.

The mRNA was hybridised to the probe overnight at 42° C. andsubsequently digested with five units of “RNAse one” (Promega) for threehours at 25° C. (Aco1) or 30° C. (Aco2 and Aco3), essentially asdescribed in Brewer et al, 1992, Promega Notes No. 38: 1-7. Productswere separated through denaturing 5% PAGE gels and visualised byautoradiography. The gels were dried and exposed to Kodak x-omat film at−70° C. with two intensifying screens for a given period described foreach gel. The signal from the gel was quantified using the AmbisRadioanalytical Scanning System.

The ribonuclease protection assay is more likely to detect low abundancemessage as it is possible to use more RNA that can be separated throughan agarose gel and blot. In addition, the hybridisation is carried outin a small volume in solution, so that the target RNA and the probe aremore concentrated. Hybridisation in solution also means that all the RNAwill be available to bind with the probe. The ribonuclease digests anysequences that do not completely hybridise which gives less chance ofmismatch hybridisation.

(b) Results

The data show that the Aco genes of tomato are highly inducible andexhibit differential expression in various tissues at different stagesof development during tomato fruit ripening. It appears that in mosttissues so far analysed at least two Aco genes are expressed (forexample, poster by C S Barry et al, ISPMB conference, Amsterdam, Jun.19-24 1994).

Aco1 appears to be the most abundantly expressed of the three genes inripening fruit where it accumulates to high levels, probably due to theautocatalytic nature of ethylene biosynthesis in this organ. Expressionshows a high induction at breaker stage and persists throughout theripening process until 12 days post breaker and probably beyond.

Expression of Aco3 is undetectable in green fruit but is induced andpeaks at the breaker stage and persists throughout ripening like Aco1but is approximately fifty times less abundant.

All three Aco genes are expressed during flower development. Larson etal (1993, pp 112-122 in Plant signals and interactions with otherorganisms, eds Raskin and Schultz, Am Soc Plant Physiologists) haveproposed a model for interorgan signaling in pollinated carnationflowers involving three distinct postpollination events that result inethylene production. It is possible that in tomato such a signalingpathway occurs and that the three Aco genes play a regulatory role.

Aco1 is induced four fold at the beginning of flower senescence and isalso greatly induced at the onset of leaf senescence. Theseobservations, together with its high abundance in senescing (ripening)fruit, indicate a possible general role for Aco1 during plant ageing.

Aco2 has been detected in fully open and senescing flowers at stages 3-5and may represent a flower specific ACC oxidase.

Aco3 appears to show the highest levels of expression throughout flowerdevelopment.

Table 1 shows the relative abundance of ACC oxidase mRNA in variousorgans and at different stages of development. In the Table, all figuresare net counts per minute (cpm); N/D means “no message detected”; UWmeans “unwounded”; W 2 h means “2 hours after wounding”.

TABLE 1 Aco1 Aco2 Aco3 Seed Germination 0 day n/d n/d n/d 8 days 2 1 3Leaves UW 1 n/d n/d W 2h 11 n/d n/d senescent 27 n/d 12 Flowers Fullyopen 12 23 58 senescent 51 25 96 FRUIT mature green 2 n/d n/d breaker +3 108 n/d 2

The spatial expression of Aco genes in flowers was examined in moredetail. Results show that Aco1 is predominantly expressed in style,stigma and petals at the onset of petal senescence; Aco2 is mainlyexpressed in anthers when the flowers open fully; Aco3 is highlyexpressed in stipe and stigma when the flowers open and remains activethrough senescence.

EXAMPLE 3 Aco Promoter/GUS Constructs for Analysis in Transgenic Plants

In order to determine their temporal and spatial expression, thepromoter fragments of the Aco genes were fused to the GUS reporter genein DNA constructs suitable for plant transformation. GUS accumulation intransgenic plants is then monitored.

In addition to the promoter-GUS fusions, the constructs containdifferent terminators. One carries the 3′UTR of Aco3, the other carriesthe 3′UTR of the polygalacturonase gene. Both constructs were assembledin pBluescript and inserted in to pBIN19 and have been transformed intoAgrobacterium.

Tomato plants have been transformed with Aco promoter/GUS constructs andhave been analysed. GUS analysis is carried out on vegetative tissue andon mature fruiting plant material when it becomes available.

FIG. 2 is a diagram of the constructs which were used to transformtomato cotyledons. The Aco1 promoter region and fragments thereof plusthe 5′UTR were fused to GUS using the binary vector pBIN19 with apromoterless GUS casette (pBI-101), forming the constructs pAco1-1824,pAco1-396 and pAco1-123. pACO1-123 contains the fragment of the Aco1promoter which shows a high degree of homology within all three tomatoAco genes. The construct pBB35S was made as a control and contains 200bp of the 35S CaMV promoter fused to GUS.

Table 2 summarises the range of GUS expression obtained with the variousconstructs identified in FIG. 2. The construct BB35S GUS is included forcomparison: it is the GUS gene driven by the CaMV35S promoter. It alsoidentifies, representative plants for further analysis (reported below).

TABLE 2 TOMATO TRANSFORMATION Representative No. of Range of GUS plants(No. of T- Construct transformants activity DNA insertions) BB35S GUS 76: medium-high H1315 (1) activity H1327 (2) 1: no activity(rearrangement in T- DNA) Aco1-124 5 5: GUS activity BB17/1 (2, oneabove background locus?) in ripening fruit H1317 (3) Aco1-396 6 5: highactivity BB13/15 (1) 1: no activity BB4/2 (2) (rearrangement in T- DNA)Aco1-1825 8 2: high activity BB15/1 (2, one 2: medium-high locus?)activity, different BB15/5 (1) pattern, abnormal phenotype 1: very lowactivity 3: no activity

Table 3 reports analysis results of a similar nature to those in Table 2for Nicotiana transformed with the same constructs.

TABLE 3 NICOTIANA PLUMBAGINIFOLIA TRANSFORAMTION Representative No. ofRange of GUS plants (No. of T- Construct transformants activity DNAinsertions) BB35S GUS 10 3: high activity Bb10/1 (1) 7: low-mediumBb10/2 (2) activity Bb10/4 (1) Aco1-124 9 Most with activity BB9/11 (2,one above background locus?) in senescent leaves BB9/12 (1) and flowersBB9/14 (2) Aco1-396 19 14: high activity BB8/1 (1) (senescent leaf)BB8/2 (3) 3: low-medium BB20/18 (1)_(—) activity 2: no activityAco1-1825 16 10: high activity BB19/17 (1) (senescent leaf) BB19/2 (1)2: medium activity BB19/10 (2) 2: activity just above background 2: noactivity

Table 4 reports the analysis of GUS expression at various stages ofplant development, showing the variation in expression levels comparedwith that found in young leaves.

TABLE 4 Relative levels of LEAco1 and uidA mRNA and GUS activity duringplant development in two representative plants harbouring the Aco1-396(plant BB13/15) and Aco1-1825 (plant BB15/5) constructs Petiole Expandedabscission Flower Flower Construct Young leaf leaf Senescent leaf zonestage 2 stage 4 IM fruit MG fruit B + 3 fruit B + 8 fruit Aco1-396 Aco1mRNA^(a) 1 8.9 31.4 nd^(b) 2.74 9 2.1  5.7 143 12.6 GUS mRNA 1 18.2 80nd 1.5 2.9 1.25  9.5 25 4.8 GUS activity 1 109 1052 639 13.5 26.5 1.0517.7 120 54 Aco1-1825 Aco1 mRNA 1 9.2 39.5 4.47 3.7 11.1 0.97 2.6/10.8^(c) 263 59.5 GUS mRNA 1 12 78 8.5 2.8 6.7 3.2   12/54 45 8.12GUS activity 1 54 307 163 13.6 22.1 2.6 20.2/51 101 78.8 Shown arerelative levels compared to those in young leaves ^(a)mRNA levels weredetermined fromat least two independent Northern or dot blots.^(b)nd:not determined ^(c)Mature green fruit from two different stagesof maturity were harvested

Table 5 reports the induction of GUS expression in representative tomatoplants in response to stimuli, wounding, ethylene and variousinfections.

TABLE 5 Fold induction of GUS activity by wounding, ethylene treatmentand infection in representative tomato transformants Construct Aco1-396Aco1-1825 BB35S GUS Wounding Leaves/2h 2.8 ± 0.45 (4) 4.93 ± 0.9 (6)1.13 ± 0.2 (6) Fruit: IM/6h 10 12.7     MG/6h 1.9 ± 0.4 (2) 1.8 ± 0.5(2) 10 ppm ethylene leaves/6h 4.4 ± 1.94 (3) 4.26 ± 1.28 (3) 0.67 ± 0.2(2) MG fruit/6h 5 2.5 ± 0.6 (2) Seedlings, 8 das dark ± 20 μM ACC 3.9 ±0.7 (2) 3.1 ± 0.09 (2) 0.27 ± 0.08 (2) dark ± 10 ppm 1.59 ± 0.26 (2)ethylene TMV infection 2 dpi 4.9 3.4 2 dpt 9.4 5.2 uninoculated 1.8 2.1leaves 7 dpi 9.4 11 dpi Cladiosporum fulvum infection 6 dpi 1.5 2.4 14dpi 3 5 Powdery mildew infection 1 dpi 3.9 8.2 2 dpi 4.3 16.6 6 dpi 1015.6 Methyl jasmonate 6h 10 μM 3.5   100 μM 6.7 4.5 5 mM α- aminobutyricacid 6h 5.9 ± 0.9 (2) 4.7 ±0.99 (2) 1.05 ±0.18 (2)

Table 6 reports the expression of GUS in germinating tomato seeds andseedlings in light and dark.

TABLE 6 GUS activity in germinating tomato seeds and seedlings Construct(plant) BB35S-GUS Aco1-396 Aco1-1825 Germination (H1327) (BB13/15)(BB15/5) Light dpi: 0 33.3* 2 27.8 4 38.2 8 23.6 38.9 14 4248 58 Light +20 μM 125.8 ACC 8 dpi Dark 8 dpi 6.4 29 10 dpi 2954 9.6 49 Dark + 10 ppmC₂H₄ 8 dpi 38.5 10 dpi 90.6 Dark + ACC + 2 mM Ag(S₂O₃)₂ 8 dpi 121 10 dpi183 Dark + 2 mM 105 Ag(S₂O₃)₂ 8 dpi Light + 3 mM 120 Ag(S₂O₃)₂ 8 dpi*GUS activity in pmol/min/mg protein Seeds were germinated on 0.7% MSagar (supplemented with 3% sucrose and 50 μg/ml kanamycin), ACC andAg(S₂O₃)₂ were included in the agar. Ethylene was injected daily intoairtight jars (390 ml) to the final concentration of 10 ppm.

7 1925 base pairs nucleic acid single linear DNA (genomic) ACO1 PROMOTER1 AGTGCTGATT ACAACATTGA AATTCTAAAT TTAGAATTTA ATATTTATTA AATGTTAGTG 60CATTTATACA AATAACATAT TACATCTCAA ATAATATTGA GTTTGTTAGA TTTTATTTGC 120CCTGATTTCT TATCATAAAT AGGTTTTCCT TTTAGGAAAA GGTTTTGAAT TGACTATTCT 180TTTTTTGGTA GGAAAAAGTT TAGGACTCTA TAAATAGAGG CATGTTCCTT CTAACTTAAT 240TAGCATTCAC AATGTAGTTT TAAGGGCTTT GAGAGTTTTG GTTAGAGGGA GAATTTGTGA 300ACCTCTCATG TATTCCGAGT GAATTGGTTG AGGTTGTTTC CCTCTGTATT TTGTACTCTC 360ATGTTTATAG TGGATTGCTC ATTTCCTTTG TGGACGTAGG TCGATTGACC GAACCACGTT 420AAATTTTTGT GTCTTTTGGT ATATTTCCTG TTCTTCTTAC TCGTGGTCTT TCGAGGTTTG 480CTTTGCTAGC TTCCGCGTTT ACACCTGCTT ATTTTCGGTC CTAACAAGTG GTATCAGAGC 540CAGATTCAAT AATGGAGTCA GGTGTAGTGG TTCGATAATC GATGATTGAA CCAAGTTAGA 600AAGAGGTGTT CATCTTGACG GGTGTAGTTC TAGCCGCAAC CTTTTTGACA GTAATGAAGA 660TTTTGATGGA GAAATTGTTT CAGAGAGGTT CTCTGTGTTG AGACATAAAT TTTGTAAAGG 720AGATTATGGA GAGGAGAAGC AAGTTGTTGA AGATTAAGTA AAGAAGGTGG ACAAATCTAT 780TTTGTCAGAA ATTCAGGCCA AGGGGGAGAT TTGTTGGGTT TTATTTGCCC TGATTTTTTA 840CCATAAATAG GTTTTCCTTT AAGGAAAAGG TTTTGAATTG ACTATTCTTT TTTTGGTAGG 900AAAAGGTTTA GGATTCTATA AATAGAGGCA TGTTCCTTCT AACTTAATTA GCATTCACAA 960TGTAGTTTTA AGGGCTTTGA GAGTTTTGGT TAGAGGGAGA ATTTGTGAAC CTCTCATGTA 1020TTCCGAGTGA ATTGGTTGAG GTTGTTTCCC TCTGTATTTT GTACTCTCAT GTTTATAGTG 1080GATTGCTCAT TTCCTTTGTG GACGTAGGTC GATTGACCGA ACCACGTTAA ATCTTTGTGT 1140CTTTTGGTAT ATTTCTCGTT GTCTTCTTAC TCGTGGTCTT TCGAGGTTTG CTTTGCTAGC 1200TTCCGCGTTT ACACCTGCTT ATTTGCGGTC CTAACAGAGT TCGATGGGTT GAATCTATAA 1260AAAGAAAAAT ATACTCGTGA TTCACGATTA TTTATATGAA AATATAATAA ATATTGAATT 1320TCCTTTGCTA TTTCTTATGT TTACGTCTTT ATATTTCAAA TTATTCCACC AATACTGACA 1380AGCCCTAGGC CATCTCTAGG AAATTCATAC AATTTTTTTT TTGTTGTTAA CTAGTTAAAT 1440TGGCAGCCTT AAAGATTATT GTAAAATTCA AGGCAACTTC CTCAAGTACT ACAACTACAT 1500TGTAACATCC CAGTCAAAGT GTCCTAAAAT TTTATAAAAT TTGACACATG AAACAATAGC 1560ACAATAAATT TTAGTACTAT TGCAGCCATG GCCCATAAGC CATCATGTAT TATAGTCAAA 1620ATGGGTCCTT TTCCAATTTG TCTTGATCCC AAAATCCCTT TGTAGGTAAG ATGGTTCAAC 1680AAGGAACTAT GACTCTTAAG GTAGACTTGG ACTCATAGAC TTGTCATAAC TCATAAAGAC 1740TTGGAATATA ATAATTATTC ATTTAAATTA TAATTCTCTA CTTTAATATC TTCTACTATA 1800AATACCCTTT CAAAGCCTCA TTATTTGTAC ATCAAACATT GATATTCATC TCTTCAATCT 1860TTTGTATTCA CATATTCTAT TTATTCAATA CACTTAGGAA AACACTTTAC CAAGAAATTA 1920AGATG 1925 2510 base pairs nucleic acid single linear DNA (genomic) ACO2PROMOTER 2 GAATTCAGAA TTTTATTTTT TTATCTCCTG ATCGAATCTG TTTACACTAAAATTACTCGT 60 TAATAGTTGA TTAAATTGGA TAAATTCATA CTTGAAAATA AAAGGTTTCGGTGATGGAGG 120 GAAATGACAG ATCTAAAAGT TTGTTTACTA GTTAACGTGA AACCAGTAATTTTTGTTTGG 180 ATCTTAGATC TATTAAATAG TTACACACAT TTTAATATTA ACCATATTGTGGTACTAATG 240 TCGTTCTAGA TTAATACGTA ATCGAGTATT GTCTAATCTC CCTTGTTACTAATTACTCGC 300 ATACGTTTAC TTCCACTTAT GTATCTTATG TATTTTATTG CTACATACTATTTTCTTCTC 360 TTCATTATAT CCAGCATGAC TGATTTACTA TTATATTTTT CTATTTACTATTATATTTTT 420 CTTTCTCATA CTTGATTTTA TTAATGATTT ACTATGATCT ATCAATAACAACTTATTTAC 480 CTTCAAAAGA TATAATCAAG ACTGTGTACA TATCAATCAC TTGATATATTTTTACTCGTT 540 AATATTATTT TTTTAATCAT CGCAACTAAC ATACTTATTG ACTAATAAAATATAAATGAC 600 TTTTCACACT TGTTTGAAGC CATATAAGTT TTTCTTCGAT CTACATCGATATAAGTTCTT 660 GGTCAAAGAT TGCATATTCT GGATATATGC TTTGTAATTA AGAAAAAAAAGGGAATTAGT 720 TAAGATAAAT TTCTACAATT ATTATACAAA AGTTAGGTTA GGTTGATGAAAAAGTGTATG 780 ACAAAGCAAA AATAAAAAAT AAAACTATGA TATGTTCAAA ATTCAAATATTTAGTCAAGA 840 ATAGTTACTT AGAATTTAAT TGGATTAAAT GAAATAATTA AAAAACTCGTGGTTCTTATG 900 TCTAACAAAA AAATCATGTT GCCAACTTAT ATTTAATGTA TTGCCTAACAAAAGTAATTC 960 GGGCCGAACG GACAAGATCT TAATTAAATT GACTTTTTAA AAATTTGAAACACGCACAAA 1020 ATTAATGTTT TTTTCGATCA AAAAGTAAAA ATACTAATTC TATATCAACCGTTCACTATA 1080 AAATTCCACT AACAATCAAC TTTTTTGTTT TGAAATCAAT TTTGTTTATCATTCTATTTC 1140 ATATTTTTTT TAAAATAACA ATATTTTTAT TAATAATAGT TAAATAATATTCAAACAAAC 1200 GTTATCGTTA CAGGGTTTTT GACTATATTA AAGAAACTTC CATGGAGCAAATGTGCAGCC 1260 CTAAAAATGT GAATTGTGTG TTAACTTCTA AATAGTATCC TTTTGTCAAATTGAACCAAA 1320 CATTTTATAA TGACACATGA AAAAAAAAAA TTAAACAAAA AAATTTAGTCAAATTGATCA 1380 AAATTTAACC ACTAGAAAAT GGGTCCAAAT TCTAATTGTC CCAACTCTAATGGGGTAGAT 1440 CAGAAGGCTA TTGGAAGATT ACTAGGTATA TGTCACTTTC GATCGGTATAAATATTGAAT 1500 AATTTTATCG ATTTAGCATT TAAATAAGAG CAAAAATAAA GTGTTTGGCAGAATTCGATG 1560 GCCTAATTTA AATTTTATAT TTATCTTAAA AAGCTCATCG AACGTATTTTAAAAACTAAA 1620 TAATTTAATA AACTAAGATA TTTCCCTTAG TTAGAAGGAT TGAGCAAAAGGTATGATTGT 1680 GGGTCAGATC AATCCATTCC GTTCTTCAGG GAAGATCTAT GGAATAGGAAAGCCAAAACG 1740 GATCTATCAA AACAGATCTA TTCTAAGTAA ATACTTGGTC GATACGAGACTCTTTCTTAG 1800 TTCAGTGGTG TTTGAAAGCA GTCTACAACG AATCAAGCAG GTCCAGTAACAACGGATAAC 1860 GGTCCTCACC GCGTTACCGA GGACCTCGTT TCAAAAAAAT ACGACGCCTGGGGGCTTTAC 1920 CAGGACTAAC TAATAAAAAG CCTAGGACCG GAAGTGATCT TAGAAACCAATCGCGTTTCG 1980 GTAAAAAACC TCAATATCGT ATTCGGATAC ATTTTCATCT TAAAAATATAATTTTTCGAC 2040 GAACATAATT CAATTGAACC ACATGTATCT AGCTTCCTCT TTAAGCTTAAGCAGATGAAA 2100 CAAGAACTTA AAAAAATAAT TATGTAATTT TCGTTATCTA TATTAAAGTTAAACTAAACA 2160 TAAATTTACC CAAAAAAAAA TTTATAATAA ATATAAAGTA ATCCCCTATAAAGTGATTAC 2220 ATATTGAGAA CCCAAAATTA TTATATTTCT ACTGAAATTT AACTTTTATTAGTTAATCCA 2280 TTGGCCACAA CCTAGTGTGG AATACCACTA TTCAATTATT ATATATTCCCTACTAGCTAT 2340 ATATGATTTA TTTCCCTATA AATACCCAAA CAAAGCCTCA ATCTTTTACACACACACCAA 2400 AAAAAGAAAA CTCACTTTCA ATATCTTCCA TCTTTTTATT CCACACACTATTTACTCTAA 2460 AAAAGAAAAA AAAAACATTT TCTTCTATTT CTTCAAGAAA TTAAAAAATG2510 2483 base pairs nucleic acid single linear DNA (genomic) ACO3PROMOTER 3 GTCGACCTGC GGATCAACGG ATCAAATTAA TTTGATATTT TAAATTAAAATTTTGAATAT 60 TAAAAAACTA TACGAAAAGT ACTATACTGT AATTTTTTTT TACAGATTAATATGATGAAA 120 AGATACATAG TAAAATATTA GTCAAAGTTC TTATAGTTTG ACTCTAAAAAAAGAAAATCG 180 TGATAATTAA AAGTTGACGA AGGGAATAAT TAGTTTGTTC TAGCCTGCATATTTCATCAG 240 CTTAGATTTT ATTAGCTGTA CGAAATTCAA CGATTAGTTA ATTAGGTAAAGTTGTTGATT 300 AACAATTAAA TGGAATGACA TAGCTAAAGT AACAAAAATA TAAAATATGATACGTCGAAA 360 ATTCAAACGT CCTAGTCAAA GATAATTAAT TAAGCCTAGA TTTGGCTTAAGAATATAATA 420 ACTAGAACTC GTGATTGCAG ACTCTATAAT TACCTCAAAT TCATTGTTAATTTTTTAAAA 480 TGTCAAATGC ATTTCCAATG ATCACATGGC CGCCCTCAAA GAAAATGACTTCAATAACAA 540 AAAATAACAT TAATAGTAAA TAAATTAATG AATTGTTATT TTAAAAAATCAATTAAGAGT 600 GGTTAGCGTA ATATACTAGG AAATTTTATA TGAATCTCAT AGTGTTAAAAGTTAATTACA 660 TCATATTTTT ATTCTTTTTT AAATTTCAAA AATTCTTTAA ATTTTGTGGAATTCGAATAT 720 ATTCCAAAAC ATTTAGATAC ATCGCGTCCA ATTTCAGATA TATTGTTCAACCTTTTGATA 780 CATCACATTT TCATTTTAGA CACAACACTC AACCGGTTCA TTTTAGATACATCACTCAGC 840 GTCCGGATAC ATCGCGTTTA GAAATGTATC CGGCTGAATC AACGCATAAAGTGATGTATT 900 ACGTCCCGAT ACATCGCGTA TAGTGATGAG TCAGACCGTA TCCAATTGATATATCGCATA 960 AACTGATGTA TTACGTCCTG ATATATACAT CATGTAAAAG GTGATGTATTCATGAATACG 1020 AGAGATTAGG GTATGTGTAA CTTTTTCAAG TTATAAATTT TTTTTAGAGAATATGATAAA 1080 ATAAAATTAA TGTAATTTAA TTGGTTAATT TTTCCGGATT TGATTTAAAAAAAAAATACA 1140 AGAGAGAGTA TAGTGATGAA GCGGAATCTT AGGGAAGATT TCTAAAATTATGTCTCTTTT 1200 TTTATGATTG TAATATCGAG TAGCTCACAA GCCTCAATTG CAACTTCATCAGTATTTGTT 1260 ATCTCCTATC ATGTACTAAG TACTAAGTAA CGTTTCTCAT TTAGAGTTAGAAAAATAAAA 1320 AGCAATCACC TAGTGTTTCC GTTCAATTAA AAGATAGCTT CTACGGCCGTATGTTTTAGC 1380 AAAACTTTTA GTTTCATTAA CTCGGGAAAA ATTTAGAAGA CATGGAAGTTCTGCACTAAA 1440 TTGCACTACA ATTTGTGTAA CAAGAAAAAA TTAATCAAGT CAACGGATAGAAATTTCATA 1500 TGAAAGATAT ATGGGGAGCG TTAAGATAGA TTCGACTGAA CTCAATAATTTTAGTTCAAA 1560 TCATGCATTT TATTTTAGAA TTTTATTTGA ATACATACAA ATAATTAATTCAGAACCAGT 1620 AATCTAAAAA GATGAGAACC GAGACTCAAT AAGGTTCAAG TCTTAGCTTGGCGCCTATCG 1680 TGCAATGTGC TAAGTACAAT CATGCACTTG ATTGAATTTA CTTAGAAAATTAGGGAACGA 1740 TTTCTCACTT AAAATACATT TTTTCCTTCT TTTCTTTGAT GATGCACTCATATTCTAAAA 1800 ATTTTAGATT CAGTCACGTC CGACATTAAA AACTTTCAAG TGTGAGGCAACTTGGTCCGA 1860 CTCTGAACCA AGAAATTTAT AATAACAGAT GAAACAATAT CATCAAATTAGCTTTCACTA 1920 GAGCAGCCTC TTCAGACAAA GCCCTTTTTA TATTAAATAT ATATATACAGGTCAAGATAA 1980 AAGGGAAAAT CATTTTTAGC CACCAAAAAA TTGGTCATTT TCCAATAGTACATCTTTAAT 2040 TAGCTCCAAA ATTAATCCCA CTTGTGTTAG GGTAAGAAGT GTCCAATAAGTAACTGTGAC 2100 CAGAATTCTA TAGCACCAGC TACACTTATG CTCCGGCTCG TATGTGTGGATGTGAGCGGA 2160 TACATTCACA CAGGAACAGC TATGACATGA TACGAATTAA TACGACTCACTATAGGAATC 2220 TGTAAGTGGA CTTGACTTTG GGGGTTTGGG ACTTGGGTGG GGCATTGTGAGACTTGGAAT 2280 AATGTAAAGA ATTGTAGGAC CAAAAATAAG GTAATTAAAT AGCAAAATCCCACTAGTTAT 2340 ATAATTCCTA AATTCTTGAT TTCTTCTCCT ATAAATACCC TTTCAAAGAATCACTCTTTT 2400 CTCATCAAAC ATTTTAATAT TCATCTCTTC AATCTCTTGT ATAATTCACATCATATAATT 2460 TAATTACCAA GAAAAATTAG ATG 2483 10 base pairs nucleicacid single linear DNA (genomic) TCA MOTIF 4 TCATCTTCTT 10 11 base pairsnucleic acid single linear DNA (genomic) GCCGCC MOTIF 5 TAAGAGCCGC C 1115 base pairs nucleic acid single linear DNA (genomic) H-BOX 6CCTACCNNNN NNNCT 15 15 base pairs nucleic acid single linear DNA(genomic) H-BOX 7 CATACCNNNN NNNCT 15

What is claimed is:
 1. A DNA sequence encoding a gene promoter capableof driving gene expression in plants which is selected from the groupconsisting of the Aco1 gene promoter having the sequence shown as SEQ IDNO: 1, the Aco2 gene promoter having the sequence shown as SEQ ID NO: 2,and the Aco3 gene promoter having the sequence shown as SEQ ID NO:
 3. 2.A gene construct comprising the Aco1, Aco2 or Aco3 promoter as claimedin claim
 1. 3. A gene construct comprising the Aco1, Aco2 or Aco3promoter as claimed in claim 1 operatively linked to a DNA specifying anRNA.
 4. A gene construct as claimed in claim 3, in which the said DNAencoding RNA is a gene regulating sequence defining an RNA in antisenseorientation to a target gene.
 5. A method for controlling the expressionof a DNA sequence in a plant, comprising providing a gene constructcomprising a gene promoter as claimed in claim 1 operatively linked to adownstream DNA specifying an RNA and a downstream 3′-transcriptionterminating signal, inserting said construct into a plant cell bytransformation and regenerating plants from the transformed cell,wherein when said gene promoter is activated, expression of said DNAoccurs.
 6. A method as claimed in claim 5, the said plant being a tomatoplant.
 7. A plant produced by the method of claim
 5. 8. A plant that hasbeen stably transformed with at least one of the sequences of claim 1.